The present invention relates to carbapenem antibiotics and, more particularly, to carbapenem compounds having a xcex2-orientated methyl group introduced at 1-position and 1-[(heterocyclyl)-azetidin-3-yl]thio group at the 2-position of carbapenem skeleton, and to antibacterial compositions containing the same as an active ingredient.
Since the discovery of thienamycin [U.S. Pat. No. 3,950,357; J. Am. Chem. Soc., 100, 313(1987)], there have been proposed many carbapenem antibiotic substances, and among them imipenem (INN) have been developed as a practically useful carbapenem antibiotic substance, and is widely used in clinical field.
Although the imipenem, developed at first as carbapenem antibiotic substance, exhibits wide antibacterial activities against gram positive or negative bacteria, it possesses the disadvantage that it is decomposed within a short period of time by renal dehydropeptidase (DHP) in the living body. For this reason, imipenem cannot be administered singly, and must be used in combination with a DHP inhibitor in order to control its decomposition leading to inactivation. Therefore, its formulation for clinical administration is a combination with cilastatin (INN) that is a DHP inhibitor.
An antibacterial agent preferred for practical clinical use; however, is one that alone can demonstrate antibacterial activity. Furthermore, the DHP inhibitor to be combined with the antibiotic could exert undesirable action on tissues of the living body. For these reasons, the combined use should be avoided wherever possible. Thus, there has been a growing demand for a carbapenem compound having sufficiently high degrees of both antibacterial activity and resistance to DHP.
There were proposed some carbapenem compounds of the type that could achieve the above-mentioned objectives. Such carbapenem compounds are 1-methylcarbapenem compounds in which a methyl group is introduced at the 1-position of the carbapenem skeleton, and it is reported that these carbapenem compounds are not only resistant to DHP but also more chemically stable than those having no methyl group at the 1-position of the carbapenem skeleton.
Under these circumstances, many researchers have specifically attempted to modify a side-chain substituent at 2-position of 1-methylcarbapenem compounds, and as a result, meropenem and biapenem are proposed for the carbapenem antibiotics that can be administered singly.
Although, the carbapenem compounds possess a potent antibacterial activity with a broad spectrum, it is anticipated that the resistant strain, which is a problem in the field of xcex2-lactam antibiotics, will appear. That is, it is well anticipated for new carbapenem antibiotics to be effective at first, but long time clinical use of it will gradually cause the resistant to appear. Therefore, there always is constant demand for development of new compounds having excellent antibacterial activity in the antibacterial field.
Under these circumstances, it is the purpose of the present invention to provide new carbapenem compounds having high antibacterial activities and a strong action of inhibiting xcex2-lactamase as well as improved resistance to DHP.
Accordingly, one subject of the present invention is to provide carbapenem compounds represented by the following formula (I): 
wherein
R is hydrogen atom, or substituted or unsubstituted lower alkyl group; and,
n is an integer of 1 or 2,
or a pharmaceutically acceptable salt thereof.
Another subject of the present invention is to provide ester compounds of formula (I), in which carboxylic acid group at 3-position is converted to ester form by various kinds of ester residues. These ester residues may be conventional ester residues, which are widely used for ester moieties for carboxylic group in this field.
The carbapenem compounds according to the present invention are characterized in that the substituent at the 2-position of 1-methyl carbapenem skeleton is specific 1-heterocyclyl-azetidin-3-ylthio group, which is never demonstrated for substituent for 2-position, and have superior antibacterial activities.
Therefore, still another subject of the present invention is to provide an antibacterial agent containing the carbapenem compounds represented by the formula (I) above, ester thereof or pharmaceutically acceptable salt thereof claimed in claim 1 as an active ingredient.
The present invention is described in more detail in the following. The terms used throughout the present specification have following meanings.
The term xe2x80x9clowerxe2x80x9d qualifying a group or a compound means that the group or the compound qualified has 1 to 7, preferably 1 to 4, carbon atoms.
The term xe2x80x9clower alkyl groupxe2x80x9d stands for a linear or branched alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl and so on.
Substituents, which can substitute to said lower alkyl group, are hydroxy group; lower alkoxy group; halogen atom such as chlorine, bromine, and so on; mono-substituted amino group; di-substituted amino group such as dimethylamino, diethylamino, methylethylamino, and the like; phenyl; substituted phenyl such as hydroxyphenyl, methylphenyl, dimethylphenyl, aminophenyl, aminomethylphenyl, nitrophenyl, and so on; nitro group; acetyl group, and the like.
When the carbapenem compounds of the present invention have an asymmetric carbon atom at the side chain of the 2-position, the isomers can be stereo-selectively obtained by using optically active starting materials, and each isomer can be isolated from the stereoisomeric mixture by usual method. Therefore, each isomer per se, as well as the stereoisomeric mixture, should be included in the compounds of the present invention.
The following are the typical carbapenem compounds of the present invention.
(1R,5S,6S)-2-[1-(1,3-imidazolin-2-yl)azetidin-3-yl]thio-6-[(R)-1-hydroxyethyl]-1-methylcarbapen-2-em-3-carbolic acid;
(1R,5S,6S)-2-[1-(1-methy-2-imidazolin-2-yl)azetidin-3-yl]-thio -6-[(R)-1-hydroxyethyl]-1-methylcarbapen-2-em-3-carbolic acid;
(1R,5S,6S)-2-[1-(1,4,5,6-tetrahydropyrimidin-2-yl)azetidin-3-yl]thio -6-[(R)-1-hydroxyethyl]-1-methylcarbapen-2-em-3-carbolic acid;
(1R,5S,6S)-2-[1-(1-methyl-1,4,5,6-tetrahydropyrimidin-2-yl) azetidin-3-yl]thio-6-[(R)-1-hydroxyethyl]-1-methylcarbapen-2-em-3-carbolic acid;
(1R,5S,6S)-2-[1-(1-(4-aminomethylbenzyl)-2-imidazolin-2-yl) azetidin-3-yl]thio-6[-(R)-1-hydroxyethyl]-1-methylcarbapen-2-em-3-carbolic acid.
Examples of the pharmaceutically acceptable salt of carbapenem compound of the present invention include non-toxic inorganic or organic salts thereof. The inorganic salts may include alkali metal salt such as sodium, potassium and the like; alkali earth metal salt such as calcium, magnesium and the like; and ammonium salt thereof. The organic salts may include triethylamine, pyridine, picoline, ethanolamine, triethanolamine, dicyclohexyl amine, N,Nxe2x80x2-dibenzyl-ethylenediamine and the like.
Furthermore, the carbapenem compounds of the present invention may be converted into a pharmaceutically acceptable acid addition salt thereof with organic or inorganic acids. Examples of inorganic acids include hydrochloric acid, nitric acid, hydrobromic acid, sulfuric acid and phosphoric acid. Examples of organic acids include organic acid such as formic acid, acetic acid, trifluoroacetic acid, maleic acid, tartaric acid, methanesulfonic acid, benzenesulfonic acid and so on; and basic or acidic amino acid such as arginine, aspartic acid, glutamic acid and the like.
The ester compound of the present invention may include ester compound prepared by the ester residue mentioned later.
The carbapenem compound of the present invention may be prepared in accordance with the processes as illustrated by the reaction scheme A shown below. 
wherein Ra is acyl group; R1 is carboxyl protecting group; and R and n have the same meanings as above.
The term xe2x80x9cacyl groupxe2x80x9d represented by Ra may be, in a narrow sense, a moiety obtained by removing the hydroxyl group from the carboxyl group of an organic carboxylic acid as well as, in a broader sense, any acyl group derived from an organic sulfonic acid or an organic phosphoric acid. Such an acyl group may include, for example, a lower alkanoyl group such as acetyl, propionyl, butyryl or the like; a (halo) lower alkyl sulfonyl group such as methanesulfonyl, trifluoromethanesulfonyl or the like; a substituted or unsubstituted arylsulfonyl group such as benzenesulfonyl, p-nitrobenzenesulfonyl, p-bromobenzenesulfonyl, toluenesulfonyl, 2,4,6-triisopropylbenzenesulfonyl or the like; and diphenylphosphoryl.
The term xe2x80x9ccaroboxyl protecting groupxe2x80x9d represented by R1 may stands for ester group. Examples include lower alkyl ester such as methyl ester, ethyl ester, n-propyl ester, isopropyl ester, n-, iso- or tert-butyl ester, n-hexyl ester or the like; aralkyl ester such as benzyl ester, p-nitrobenzyl ester, o-nitrobenzyl ester, 2,4-dinitrobenzyl ester, p-chlorobenzyl ester, p-bromobenzyl ester, p-methoxybenzyl ester or the like; lower aliphatic acyloxyalkyl ester such as acetoxymethyl ester, acetoxyethyl ester, propionyloxymethyl ester, n- or iso-butyryloxymethyl ester or the like.
According to Process (a) of the reaction scheme (A), the compound of formula (IV) is obtained by reacting the compound (II) with the compound (III).
The reaction of the compound (II) with the compound (III) may be carried out, for instance, by reacting the compound (II) with approximately 0.5 molar to approximately 5 molar, preferably from approximately 0.8 molar to approximately 3 molar in amount of the compound (III) in an appropriate solvent such as tetrahydrofuran, dichloromethane, dioxane, dimethylformamide, dimethylsulfoxide, acetonitrile, hexamethylphosphoramide or the like, preferable in the presence of a base such as sodium hydrogen carbonate, potassium carbonate, triethylamine, diisopropylethylamine, especially diisopropylethylamine, at a temperature ranging from approximately xe2x88x9240xc2x0 C. to 25xc2x0 C., preferably under ice-cooling for approximately 30 minutes to 24 hours.
Although it is not required, this reaction is preferred to be carried out in an inert atmosphere, for example in an atmosphere of nitrogen gas or argon gas.
The reaction described above provides the resulting compound of formula (IV), and the resultant may be used for the next reaction without further purification; or the compound (IV) may be isolated and purified in conventional manner such as by means of filtration, extraction, washing, removal of the solvent, drying, column chromatography and so on.
Next, according to Process (b), the compound of the formula (IV) obtained by Process (a) described above is converted to the carbapenem compound of the present invention of the formula (I) by the removal reaction of the carboxyl protecting group known per se, such as solvolysis or hydrogenolysis.
In a typical reaction, the compound of the formula (IV) may be treated in a mixture solvent having pH ranging from 5 to 7, using hydrogen under approximately 1 to 5 atmospheric pressures, in the presence of a catalyst for hydrogenation such as platinum oxide, palladium-activated carbon or palladium hydroxide-activated carbon at temperature ranging from approximately 0xc2x0 C. to 50xc2x0 C. for approximately 0.25 to 5 hours. Examples of the mixture solvent used in the reaction may be an acetate buffer solution having pH ranging 5 to 7, a morpholinepropanesulfonic acid-sodium hydroxide buffer solution having pH ranging 5 to 7, or phosphoric acid buffer solution having pH ranging 5 to 7, or further containing alcoholic solvent and/or tetrahydrofuran. Furthermore, tetrahydrofuran-water, tertahydrofuran-ethanol-water, dioxane-water, dioxane-ethanol-water, n-butanol-water containing potassium phosphate dibasic, sodium phosphate dibasic or sodium bicarbonate or the like may be used for the reaction mixture solvent.
Furthermore, the removal of the protecting group R1 may also be carried out by reacting the compound of formula (IV) with zinc in a buffer solution. In typical reaction, the compound of formula (IV) may be treated with zinc in the buffer solution of pH 5 to 7 such as a phosphate buffer solution, an acetate buffer solution, morpholinepropanesulfonate buffer solution, and N-methylmorpholine buffer solution.
Zinc used in the reaction may include, for example, elemental zinc in the form of powder, flower or granule or the like. The amount of zinc used in this reaction is not strictly limited; however, it is conveniently about 1 to 10 parts by weight, preferably 1 to 5 parts by weight per part by weight of the compound of formula (IV) to be treated.
In this reaction, an organic solvent may be used in combination. Examples of the solvent are alcohols such as ethanol, propanol, n-butanol and the like; ethers such as diethylether, tetrahydrofuran, dioxane and the like; acetonitrile, dimethylformamide, dimethylacetamide and the like. Usually, the reaction may be finished in approximately 0.1 to 5 hours in a reaction temperature from about xe2x88x9220xc2x0 C. to about 50xc2x0 C., preferably from the room temperature to about 30xc2x0 C.
Furthermore, the compound of the present invention in which the group xe2x80x9cRxe2x80x9d is hydrogen atom may be prepared from the compound of formula (III) in which the group xe2x80x9cRxe2x80x9d is amino protective group, such as p-nitrobenzyloxycarbonyl group (PNZ) or tert-butoxycarbonyl group (Boc), in accordance with the condensation reaction of step (a) in Reaction Scheme A above and then, the removal reaction of the amino and carbonyl protecting groups of the resulting compounds in step (b).
The removal of both amino and carbonyl protecting groups may be preferably conducted by hydrogenolysis as already described in step (b) as mentioned above.
The carbapenem compounds of the formula (I) according to the present invention thus obtained may be isolated and purified by the conventional manner, or by using ion-exchange chromatography or synthetic absorbent polymer resin chromatography method, if necessary.
The compounds of the formula (III) used in the above procedure as the starting compounds, may be prepared in accordance with the processes as illustrated by the reaction scheme B shown below. 
wherein R2 and R3 are thiol protecting groups.
For example, the compound (VII), which may be obtained by the condensation reaction of 3-hydroxyazetidine (V) with compound (VI) having methylthio group in the molecule, is converted to the compound (VIII) under Mitsunobu""s reaction condition such as by treating with thioacetic acid. Thus, hydroxy group of the compound (VII) is converted to protected mercapto group, which is protected by thiol protecting group (acetyl group).
Then, the obtained compound (VIII) is converted to the objective compound (III) by removal of the thiol protecting group of the compound (VIII). In the process of the synthesis of the carbapehem compound of formula (I) as shown in the Reaction Scheme A, the crude compound (III) which is obtained by removal of the thiol protecting group of the compound (VIII) can be used for the reaction with compound (II) without purification.
Each process of the Reaction Scheme B may be carried out in the presence of an appropriate inert solvent or catalyst. Examples of the solvent are alcohols such as methanol, ethanol, isopropanol and the like; ethers such as diethylether, tetrahydrofuran, dioxane and the like; benzenes such as benzene, toluene, xylen and the like; acetonitrile, dimethylformamide and the like.
The detailed reaction conditions of the processes of the Reaction Scheme B are described by the Preparation Examples mentioned later, and various kinds of compounds of formula (III) may be obtained by the same manner as described in the Preparation Examples.
The carbapenem compounds of the present invention are novel compounds that are not disclosed specifically in the prior publication, and have superior antibacterial activities. The remarkably high antibacterial activities of the compounds according to the present invention have been proved by antibacterial tests described below.
[Antibacterial Tests]
1. Test Procedures
The antibacterial activities were tested by an agar plate dilution method in accordance with the standard method of The Japanese Chemotherapy Society [Chemotherapy, Vol.29, 76-79(1981)]. A Mulluer-Hinton (MH) agar liquid medium of a test microorganism was cultured overnight at 37xc2x0 C. and the resultant culture medium was diluted with a buffered saline gelatin (BSG) solution to contain approximately 106 cells of the test microorganisms per milliliter, and then the diluted solution was inoculated with a micro-planter at the rate of approximately 5 xcexcl on a MH agar medium containing a test compound. This medium was then incubated at 37xc2x0 C. for 18 hours. The minimum inhibitory concentration (MIC) is determined as a minimum concentration in which no test microorganism could grow.
It is noted here that the test organisms used were all standard strains, and test compounds were Compound (8) and (15) obtained in Examples mentioned later.
2. Results
Table 1 shows the test results.
The foregoing results clearly demonstrate that the carbapenem compounds according to the present invention have superior antibacterial activities.
Furthermore, the compounds of the present invention are characterized by having a methyl group introduced at 1 xcex2-position and specific 1-aza-3-thiabicyclo ring group at the 2-position of carbapenem skeleton, and therefore are stable against renal dehydropeptidase (DHP) and are also stable chemically and physically.
[Toxicity Tests]
Toxicological studies were carried out using a group of 10 male mice of ddY strain weighing from 20 to 23 grams. Solutions containing each of the carbapenem compounds of the present invention obtained by Examples mentioned later were administered subcutaneously to the mice and subjected to observations for one week. The results have revealed that the groups of mice to which the carbapenem compounds of the present invention had been administered in the amount of 500 mg/kg were alive without any abnormal findings.
As described above, the carbapenem compounds according to the present invention permit a single administration without combining with any other DHP inhibitor, and are extremely useful as antibacterial agents for therapy and prevention of infectious diseases from various pathogenic organisms.
The carbapenem compounds or pharmaceutically acceptable salt thereof according to the present invention may be administered as an antibacterial agent to the human being and other mammalian animals in the form of a pharmaceutically acceptable composition containing an antibacterial effective amount thereof. The administration dose may vary in a wide range with age, weight, conditions of patients, forms or route of administration, physician""s diagnoses or the like and may be orally, parenterally or topically administered. To adult patients, a standard daily dose range from approximately 200 to approximately 3,000 mg once or in several installments per day.
The pharmaceutically acceptable composition of the carbapenem compounds according to the present invention may contain an inorganic or organic, solid or liquid carrier or diluent, which is conventionally used for preparation of medicines, particularly antibiotic preparations, such as an excipient, e.g., starch, lactose, white sugar, crystalline cellulose, calcium phosphate or the like; a binder, e.g., acacia, hydroxypropyl cellulose, alginic acid, gelatin, polyvinyl pyrrolidone or the like; a lubricant, e.g., stearic acid, magnesium stearate, talc, hydrogenated plant oil or the like; a disintegrator, e.g., modified starch, calcium carboxymethyl cellulose, low substituted hydroxypropyl cellulose or the like; or a dissolution aid, e.g., a non-ionic surfactant, an anionic surfactant or the like, and may be prepared into forms suitable for oral, parenteral or topical administration.
The formulation suitable for oral administration may include solid preparations such as tablets, coatings, capsules, troches, powders, fine powders, granules, dry syrups or the like, and liquid preparations such as syrups and the like; the formulations suitable for parenteral administration may include, for example, injectable solutions, drip-feed solutions, depositories or the like; and the formulations suitable for topical administration may include, for example, ointments, tinctures, creams, gels or the like. These formulations may be formed by procedures known per se to those skilled in the art in the field of pharmaceutical formulations.
The carbapenem compounds according to the present invention are suitable for administeration in the form of parenteral formulations, particularly in the form of injectable solutions.